The present invention describes processes for measuring DNA or RNA binding proteins, specific nucleic acids, as well as enzyme activities and nucleic acid damaging activities using labeled nucleic acids or labeled protein/peptide molecules.
The ability to measure the activity or amount of an analyte in a biological sample is critical in the fields of life sciences research and medical diagnostics. A broad class of important assays are assays that measure the activity of enzymes that catalyze the synthesis or cleavage of polypeptides or polynucleotides (or, similarly, assays for substrates, products or inhibitors of these enzymes). These enzymes include proteases, nucleases, polymerases, ligases and the like. Another broad class of important assays measure the interaction of nucleic acids with proteins or other nucleic acids.
Enzymatic activity may be measured through the use of synthetic enzyme substrates that show changes in color or fluorescence when acted upon by the enzyme. This approach, however, requires the design and synthesis of a custom reagent for every enzyme; a process that can be laborious, time consuming, and expensive. In addition, it is often desirable to measure the activity of an enzyme on its natural substrate.
A possibly more generic approach for measuring protease or nuclease activity is the Scintillation Proximity Assay (SPA); see, e.g., U.S. Pat. No. 4,568,649 and Published PCT Application WO90/03844. SPA uses small microspheres that are derivatized in such a way as to bind specific molecules. If a radioactive molecule is brought into close proximity to the bead a scintillant incorporated in the microsphere is excited and subsequently emits light. Radioactive molecules not bound to the microspheres excite the scintillant to a much lesser extent than radioactive molecules bound to the beads and, therefore, produce a weaker light signal. A number of assay formats have been described using SPA detection technology including protease (Wilkinson et al., Pharm. Res. (1993) 10, 562) and ribonuclease protection assays (Kenrick, et al., Nucl. Acids Res. (1997) 25, 2947).
While SPA has proved useful for these and other classes of assays, the technique has several disadvantages. The primary problem with SPA is the requirement for radioactive reagents. Because of the severe cost, safety, environmental, and regulatory issues associated with the use of radioisotopes, there is a clear need for alternative assay techniques that do not use radioactive materials. The background signal associated with the SPA approach is relatively high due to the inability of the assay format to totally discriminate the signal that is generated from free from that generated from bound radioactivity. In addition, the sensitivity of SPA has been found to be limited; there is a need for more sensitive assay techniques. As a result of the high background signal and low to moderate sensitivity, SPA approaches generally possess relatively low signal to noise ratios which, in many cases, can adversely effect assay performance.
The most common method for measuring the specific interaction of proteins with nucleic acids is the gel shift or electrophoretic mobility shift assay. This approach has been widely used for the study of sequence-specific binding proteins, especially transcription factors. The basis for the approach is that complexes of DNA and protein have a reduced or xe2x80x9cshiftedxe2x80x9d mobility during non-denaturing gel electrophoresis. DNA duplexes, containing a specific protein binding sequence, are end labeled (generally with a radioactive label) and incubated with a sample containing the specific binding protein. The sample is subsequently analyzed by electrophoresis and the specific complexes are detected following autoradiographic analysis of exposed film. The amount of specific binding protein is determined semi-quantitatively by measuring the amount of the specific protein-DNA complex. This approach has been largely relegated to the world of basic exploratory research, primarily because of the inherent limitations of gel electrophoresis: i) the technique is complex and can usually only be carried out by highly trained lab technicians; ii) the technique is slow and laborious and is, therefore, not suited to the high throughput screening of large numbers of samples; and iii) the technique is, at best, semi-quantitative in nature. In addition, the use of radioactivity has also posed as an obstacle to some for the use of this technique. Although non-radioactive approaches have recently emerged, these approaches are accompanied by significant increases in labor.
Electrochemiluminescent Detection Technology
Numerous methods and systems have been developed for the detection and quantitation of molecules of interest in biochemical and biological samples. Methods and systems which are capable of measuring trace amounts of microorganisms, pharmaceuticals, hormones, viruses, antibodies, nucleic acids and other proteins are of great value to researchers and clinicians.
A very substantial body of art has been developed based upon binding reactions, e.g., antigen-antibody reactions, nucleic acid hybridization techniques, protein-ligand systems as well as for formats for measuring a variety of enzymatic activities. The high degree of specificity in many biochemical and biological assay systems has led to many methods and systems of value in research and diagnostics. Typically, the existence of an analyte or enzyme of interest is indicated by the presence or absence of an observable xe2x80x9clabelxe2x80x9d attached to one or more of the binding molecules or starting substrates.
Electrochemiluminescent (ECL) assays provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescence is triggered by a voltage impressed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of such ECL techniques, reference is made to U.S. Pat. No. 5,714,089, U.S. Pat. No. 5,591,581, U.S. Pat. No. 5,597,910, U.S. Pat. No. 5,679,519, PCT published application WO90/05296, PCT published application WO92/14139, PCT published application WO90/05301; PCT published application WO96/24690, PCT published application US95/03190, PCT published application WO96/06946, PCT published application WO96/33411, PCT published application WO87/06706, PCT published application WO96/39534, PCT published application WO93/10267, PCT published application WO96/41175, PCT published application WO98/12539, PCT published application WO96/28538, PCT published application WO96/21039, PCT published application WO97/33176, PCT published application WO96/17248, and PCT published application WO96/40978, and U.S. patent application Ser. No. 09/023,483. The disclosures of the aforesaid applications are hereby incorporated by reference in their entirety. Reference is also made to two reviews on ECL technology: Blackburn et al. (Clinical Chemistry, 1991, 37, 1534-1539) and a 1994 review of the analytical applications of ECL by Knight, et al. (Analyst, 1994, 119: 879-890) and the references cited therein. The disclosure of the aforesaid articles are hereby also incorporated by reference in their entirety.
It is an object of the invention to provide a simple, accurate, and reliable assay for nucleic acid damaging activity.
It is an object of the invention to provide a simple, accurate, and reliable assay for measuring nucleic acid repair proteins.
It is an object of the invention to provide a simple, accurate and reliable assay for measuring enzyme activity in a sample.
It is also an object of the invention to provide a simple, accurate and reliable assay for measuring proteins that bind nucleic acid.
It is an object of the invention to provide a simple, accurate and reliable assay for measuring inhibitors of enzyme activity in a sample.
It is an object of the invention to provide a simple, accurate and reliable assay for measuring substrates of enzymes in a sample.
It is an object of the invention to provide a simple, accurate and reliable assay for measuring specific nucleic acid sequences in a sample.
The present invention describes processes for measuring DNA or RNA binding proteins, specific nucleic acids, as well as enzyme activities and nucleic acid damaging activities using labeled nucleic acids or labeled protein/peptide molecules. As used herein, the term xe2x80x9cmeasuringxe2x80x9d or xe2x80x9cmeasurexe2x80x9d means detecting and/or quantitating.
The invention includes a method for assaying a sample for the presence of a nucleic acid repair protein, which comprises: mixing at least one predetermined single- or double-stranded nucleic acid containing at least one label and containing a nucleic acid repair protein binding domain with a sample which may contain a nucleic acid repair protein; incubating the mixture under conditions which allow the binding of said nucleic acid repair protein to said at least one predetermined single- or double-stranded nucleic acid repair protein binding domain to form a complex; adding a nucleic acid-cleaving reagent (e.g., an enzyme) to the mixture; incubating the mixture under conditions which allow the cleavage of said at least one predetermined single- or double-stranded nucleic acid which has not formed a complex; and measuring the amount of said complex to measure said nucleic acid repair protein.
The invention includes a method for assaying a sample for the presence an inhibitor of a nucleic acid repair protein, which comprises: mixing at least one predetermined single- or double-stranded nucleic acid containing at least one label and containing a nucleic acid repair protein binding domain and a predetermined nucleic acid repair protein with a sample which may contain an inhibitor of the binding of said nucleic acid repair protein with said at least one predetermined single- or double-stranded nucleic acid; incubating the mixture under conditions which allow the binding of said nucleic acid repair protein to said at least one predetermined single- or double-stranded nucleic acid to form a complex; adding a nucleic acid-cleaving enzyme or reagent to the mixture; incubating the mixture under conditions which allow the cleavage of said at least one predetermined single- or double-stranded nucleic acid which has not formed a complex; and measuring the amount of said complex to measure said inhibitor.
The invention includes a method for assaying a sample for a nucleic acid damaging activity which comprises: mixing at least one single- or double-stranded nucleic acid containing at least one electrochemiluminescent label with a sample which may contain a nucleic acid-damaging activity; incubating the mixture under conditions which allow said nucleic acid damaging activity to damage said nucleic acid: adding a reagent (e.g., an enzyme) that specifically cleaves damaged nucleic acid: incubating the mixture under conditions that allow the cleavage of damaged nucleic acid; and measuring the amount of cleaved nucleic acid to measure said nucleic acid damaging activity.
The invention includes a method for measuring an inhibitor of a nucleic acid damaging activity, which comprises: mixing at least one single- or double-stranded nucleic acid containing at least one electrochemiluminescent label with a predetermined nucleic acid damaging activity, and a sample which may contain an inhibitor of said nucleic acid damaging activity; incubating the mixture under conditions which allow said nucleic acid damaging activity to damage said nucleic acid; adding a reagent (e.g., an enzyme) that specifically cleaves damaged nucleic acid; incubating the mixture under conditions that allow the cleavage of damaged nucleic acid; and measuring the amount of cleaved nucleic acid to measure said inhibitor of said nucleic acid damaging activity.
The invention includes a kit comprising in one or more containers: a nucleic acid having a predetermined nucleic acid repair protein binding domain wherein said nucleic acid has a detectable moiety attached thereto; and a nucleic acid-cleaving enzyme or nucleic acid-cleaving reagent.
The invention includes a nucleic acid comprising a predetermined nucleic acid repair protein binding domain wherein said nucleic acid has a detectable moiety attached thereto.
This invention includes a method for measuring the amount or activity of an enzyme in a sample that catalyzes the cleavage of a molecule into two or more products, the method comprising the following steps: i) mixing a sample which may contain the enzyme with a substrate of the enzyme, an ECL label, and a solid phase, wherein the substrate is linked to the ECL label and is linked or capable of being linked to the solid phase and wherein the enzyme is capable of cleaving the substrate to form at least one product that is linked to an ECL label but that is not linked or capable of being linked to the solid phase; ii) inducing the mixture to emit electrochemiluminescence and iii) measuring the electrochemiluminescence so as to measure the amount or activity of the enzyme. The invention also includes analogous methods for measuring the amount or activity of substrates or inhibitors of enzymes that catalyze the cleavage of an enzyme into two or more products.
This invention includes a method for measuring the amount or activity of an enzyme that catalyzes the joining of two or more substrates to form a product, the method comprising the following steps: i) mixing a sample which may contain the enzyme with two substrates of the enzyme, an ECL label, and a solid phase, wherein one of the substrates is linked or capable of being linked to the solid phase and another of the substrates is not linked or capable of being linked to the solid phase but is linked to the ECL label and wherein the enzyme is capable of forming a product that is linked to the ECL label and linked or capable of being linked to the solid phase; ii) inducing the mixture to emit electrochemiluminescence and iii) measuring the electrochemiluminescence so as to measure the activity or amount of the enzyme. The invention also includes analogous methods for measuring the amount or activity of substrates or inhibitors of enzymes that catalyze the joining of two or more substrates to form a product.
This invention includes a method for measuring enzyme activities that cleave nucleic acid molecules in a sample, which comprises, mixing at least one or more single- or double-stranded nucleic acid molecules containing one or more ECL labels, adding a sample which may contain a nucleic acid-cleaving enzyme, and incubating under conditions which allow cleavage of the nucleic acid sequence, contacting this mixture with at least one solid phase, preferentially inducing ECL from ECL labels in solution or on the solid phase and measuring the ECL emission so as to measure the amount of cleaving activity in the sample. This invention includes a method for detecting and/or quantitating enzyme activities that cleave peptide or protein molecules in a sample, which comprises, mixing at least one or more peptide or protein molecules containing one or more ECL labels, contacting this mixture with at least one solid phase, adding a sample which may contain a peptide- or protein-cleaving enzyme, inducing ECL from ECL labels in solution and/or on the solid phase and measuring the ECL emission so as to measure the amount of cleaving activity in the sample.
This invention includes a method for measuring enzyme activities that covalently join nucleic acid molecules in a sample, which comprises, mixing at least one or more single- or double-stranded nucleic acid molecules containing one or more ECL labels, adding a sample which may contain a nucleic acid-joining enzyme, incubating under conditions which allow the joining of the nucleic acid sequences, contacting this mixture with at least one solid phase, inducing ECL from ECL labels in solution and/or on the solid phase and measuring the ECL emission so as to measure the amount of joining activity in the sample.
This invention includes a method for measuring enzyme activities that covalently join nucleic acid molecules in a sample, which comprises, mixing at least one or more single- or double-stranded nucleic acid molecules containing one or more ECL labels, contacting one or more of the labeled nucleic acid molecules with at least one solid phase, adding a sample which may contain a nucleic acid-joining enzyme, incubating under conditions which allow the joining of the nucleic acid sequences, inducing ECL from ECL labels in solution and/or on the solid phase and measuring the ECL emission so as to measure the amount of joining activity in the sample.
This invention includes a method for measuring nucleic acid binding proteins in a sample, which comprises the following steps: i) contacting the sample with one or more single- and/or double-stranded nucleic acid molecules containing a specific protein binding nucleotide sequence; ii) incubating under conditions which allow the specific binding of the nucleic acid binding proteins to the protein binding nucleotide sequence; iii) adding a nucleic acid cleaving reagent or enzyme; iv) incubating the sample under conditions that allow for the cleavage of the nucleic acid; and v) measuring the extent of nucleic acid cleavage.
This invention includes a method for detecting and/or quantitating nucleic acid binding proteins in a sample, which comprises, mixing at least one or more single- or double-stranded nucleic acid molecules containing a specific protein binding nucleotide sequence and containing one or more labels, contacting one or more of the labeled nucleic acid molecules with at least one solid phase, adding a sample which may contain one or more nucleic acid binding proteins, and incubating under conditions which allow the specific binding of the proteins to the protein binding nucleotide sequence, adding a nucleic acid cleaving reagent or enzyme, and incubating the sample under conditions that allow for the cleavage of the nucleic acid molecules, and measuring the amount of labeled nucleic acid on the solid phase and/or in the solution phase.
This invention includes a method for measuring nucleic acid binding proteins in a sample, which comprises the following steps: i) contacting the sample with one or more single- and/or double-stranded nucleic acid molecules containing a specific protein binding nucleotide sequence and containing a number of modified nucleotides that are resistant to nuclease digestion; ii) incubating under conditions which allow the specific binding of the proteins to the protein binding nucleotide sequence; iii) adding a nucleic acid cleaving reagent or enzyme; iv) incubating the sample under conditions that allow for the cleavage of the nucleic acid molecules; v) and measuring the extent of nucleic acid cleavage.
This invention includes a method for measuring nucleic acid binding proteins in a sample, which comprises the following steps: i) mixing at least one or more single- or double-stranded nucleic acid molecules containing a specific protein binding nucleotide sequence and containing a number of modified nucleotides that are resistant to nuclease digestion, and containing one or more labels; ii) contacting one or more of the labeled nucleic acid molecules with at least one solid phase; iii) adding a sample which may contain one or more nucleic acid binding proteins, and incubating under conditions which allow the specific binding of the proteins to the labeled nucleic acid sequences; iv) adding a nucleic acid cleaving reagent or enzyme, and incubating the sample under conditions that allow for the cleavage of the nucleic acid molecules, and measuring the amount of label on the solid phase and/or in solution.
This invention includes a method for measuring nucleic acid binding proteins in a sample, which comprises the following steps: mixing at least one or more single- or double-stranded nucleic acid molecules containing a specific protein binding nucleotide sequence, and containing a number of modified nucleotides that are resistant to nuclease digestion, and containing one or more labels; adding a sample which may contain one or more nucleic acid binding proteins, and incubating under conditions which allow the specific binding of the proteins to the labeled nucleic acid sequences; adding a nucleic acid cleaving reagent or enzyme, and incubating the sample under conditions that allow for the cleavage of the nucleic acid molecules; contacting one or more of the labeled nucleic acid molecules with at least one solid; and measuring the amount of labeled nucleic acid on the solid phase and/or in solution.
This invention includes a method for measuring nucleic acid binding proteins in a sample, which comprises the following steps: mixing at least one or more single- or double-stranded nucleic acid molecules containing a specific protein binding nucleotide, and containing a number of modified nucleotides that are resistant to nuclease digestion, and containing one or more labels; contacting one or more of the labeled nucleic acid molecules with at least one solid phase; adding a sample which may contain one or more nucleic acid binding proteins, and incubating under conditions which allow the specific binding of the proteins to the labeled nucleic acid sequences; adding a nucleic acid cleaving reagent or enzyme, and incubating the sample under conditions that allow for the cleavage of the nucleic acid molecules; and measuring the amount of labeled nucleic acid on the solid phase and/or in solution.
This invention includes a method for measuring specific nucleic acid sequences in a sample, which comprises, mixing at least one or more predetermined single-stranded nucleic acid molecules containing one or more ECL labels, contacting one or more of the labeled nucleic acid molecules with at least one solid phase, adding a sample which may contain one or more of the specific nucleic acid sequences, incubating under conditions which allow the specific binding of the sample nucleic acid sequences to the labeled nucleic acid sequences, adding a nucleic acid cleaving reagent or enzyme, incubating the sample under conditions that allow for the cleavage of the nucleic acid molecules, and detecting and/or quantitating the amount of labeled nucleic acid on the solid phase and/or in solution.
This invention also includes a method for measuring specific nucleic acid sequences in a sample, which comprises, mixing at least one or more predetermined single-stranded nucleic acid and containing one or more ECL labels, adding a sample which may contain one or more of the specific nucleic acid sequences, incubating under conditions which allow the specific binding of the sample nucleic acid sequences to the labeled nucleic acid sequences, adding a nucleic acid cleaving reagent or enzyme, incubating the sample under conditions that allow for the cleavage of the nucleic acid molecules, contacting one or more of the labeled nucleic acid molecules with at least one solid phase, measuring the amount of labeled nucleic acid on the solid phase and/or in solution.